It is well known that the mechanical properties of cells contain important information about their state and their function. For example, cancerous cells are more deformable than non-cancerous cells, and also, among cancerous cells, metastatic cancerous cells are more deformable than less aggressive cancerous cells (cf. J. Guck et al., “Optical Deformability as an Inherent Cell Marker for Testing Malignant Transformation and Metastatic Competence”, Biophys. J., vol. 88:5, p. 3689-3698, May 2005).
It follows from the above that it is of high importance to determine the mechanical properties of cells. Here, mechanical properties encompass properties such as the Young's modulus, the shear modulus, the viscosity, . . . . However, the above term is not limited to such quantities, and it also encompasses any physical quantities which describe the mechanical behavior of cells. Such a quantity could, e.g., be the relative deformation of a cell compared with its initial shape when subjected to a particular force.
Given their size, it is also evident that macroscopic methods cannot be used for determining the mechanical properties of cells. Therefore, in the prior art, several methods have been proposed for measuring these properties.
One such method is micropipette aspiration. The method of micropipette aspiration makes use of micrometer scaled pipettes to probe the mechanical properties of single cells. The invention goes back to the year 1954 when Mitchision and Swann used a device called the “cell elastimeter” to measure the membrane properties of urchin eggs which have diameters of about 200 μm, Later, smaller pipettes were used: Typical diameters range from 1 to 10 μm. The pipette is mounted on a micromanipulator inside a chamber that contains the sample. The micromanipulator can move the pipette along three axes to grab cells and manipulate them by applying a suction pressure. The sample chamber is observed using a microscope and a hydrostatic device creates a pressure difference between the chamber and the inside of the pipette. The forces that can be reproducibly generated with micropipettes range from 0.01 nN to 104 nN. The limiting factor for very small forces is the hydrostatic generation of the suction pressure. It is even sensitive to the humidity of the air. The maximal suction pressure is limited by the vapor pressure of water to a value of 96 nN/μm2. The force F acting on the cells is given by: F=ΔPπR2P where RP is the radius of the pipette tip and ΔP is the suction pressure.
A further way of determining the mechanical properties of individual cells is by using an atomic force microscope (AFM), which was developed in 1986 by Binning, Quate and Gerber. It is a descendant of scanning tunneling microscopes that relied on electrically conducting surfaces. In contrast the AFM feels the surface with a tip by mechanical forces. This makes the method appropriate to a lot of samples, such as, for example, biological tissues or single cells. Scanning tunneling microscopy and atomic force microscopy have in common that the spatial resolution does not have the limitations of a light microscope.
The central part of an AFM is the cantilever. It is basically a beam with an attached tip or, in particular for measurements of cell mechanics, a bead. The force acting on the cantilever is determined by observing the deflection of the cantilever with a reflected laser beam.
A further method for determining the mechanical properties of cells is the optical stretcher developed by Guck et al., which is described in the paper “The Optical Stretcher: a novel laser tool to micromanipulate cells”, J. Guck et al., Biophysical Journal, vol. 81, issue 2, pages 767-784, 2001. The optical stretcher makes use of the momentum transfer that happens if a light ray penetrates the surface of an object having a higher or lower refractive index than the surrounding medium.
This principle can be applied to suspended cells in optical traps, since they have a refractive index different from the surrounding medium. In the optical stretcher the cells are held in an optical trap formed by two counter-propagating high power near-infrared laser beams. These beams are exactly opposed and—in contrast to other optical traps—not focused but divergent. Since the two beams have the same intensity there is a stable trapping position in the center between the fiber ends. The principle of the optical stretcher was published in 1999 by J. Guck and J. Käs as WO 99/44488 and proved itself to be highly useful in diagnostic and scientific applications. The optically induced stress is not acting homogeneously on the cell's surface, which is the reason why a deformation of the cell results. This deformation gives an indication of their mechanical properties. Softer cells will deform more under the same stress than stiffer ones. Because the manual evaluation of deformations would be tedious and user biased, a custom automated image processing algorithm was used to quantify the deformation for further analysis.
To do so, the contour of the trapped cell has to be extracted from phase contrast images. Therefore the picture of the cell is transformed into polar coordinates taking into account their almost circular shape. In the transformed image the gradient in a radial direction gives a stable criterion for the position of the cell edge. To smoothen the contour, the contour is transformed into Fourier space, and an inverse transform is carried out whilst neglecting higher order terms. The deformation is quantified by fitting an ellipse to that contour, which allowed for obtaining the lengths of the axes of the ellipse to sub-pixel accuracy.
A further method is to make use of hydrodynamic stretching. There are several microfluidic approaches making use of that principle. Interaction with the flow of the surrounding medium causes stress on the surface of a cell. The forces originate either from a non-zero shear rate of the medium—which causes tangential shear forces—or from inertial momentum transfer—causing forces normal to the object's surface. Momentum transfer only plays a role at higher Reynolds numbers when the inertial terms in the Navier-Stokes-Equations become relevant. In contrast, shear forces are dominant at low Reynolds numbers. The idea behind hydrodynamic stretching is to create a flow field that is able to create enough stress on a cell's surface to cause measurable deformation. Such deformations were observed for red blood cells—which are relatively soft—when flowing through capillaries. However the implementation of hydrodynamic stretching for stiffer cells is challenging. Stiffer cells need higher forces and thus higher flow rates to be deformed measurably. These high velocities make it harder to acquire images of the deformed cells. Further, such approaches, one of which is disclosed in Dupire et al, “Full dynamics of a red blood cell in shear flow”, Proc. Natl. Acad. Sci. USA, vol. 109, no 51, pp. 20808-20813, 2012, only infer the cell mechanics indirectly, in this case by the tumbling motion of a cell.
D. Gossett et al., “Hydrodynamic stretching of single cells for large population mechanical phenotyping”, Proc. Natl. Acad. Sci. USA, vol. 109, no 20, pp. 7630-7635, 2012, discloses a method of obtaining mechanical properties of cells by placing them at a point where two counterpropagating liquid flows meet. Due to the momentum change at this point, the cells are distorted, and by making use of an image analysis program, the mechanical properties are evaluated.
This approach requires placing the cells at a point which is unstable due to the two counterpropagating flows. Secondly, the image analysis can only be done after the measurement, since a complicated algorithm is used. This means that it is impossible to analyse the data in real time (i.e. during the measurement), but they have to be stored as a video file only to be analysed later. A consequence of this is that the approach cannot be used for sorting cells according to their mechanical properties. This is because for such a sorting, one needs to determine the properties of a particular cell as it is being transported through the channel, since one has to take a decision as to where to send the cell immediately during the measurement. Since this is impossible in the mechanism proposed by Gossett et al., this is another disadvantage of their method.
U.S. Pat. No. 6,522,781 relates to an apparatus for analyzing particle shapes. That apparatus is not capable of determining the mechanical properties of cells. US 2007/0008528 A1 discloses an apparatus which is reported of being capable of measuring the mechanical properties of cells in real time. However, since it only makes use of an intensity variation of the incident light, the quality of its data is low.